Spatial Learning by Using Non-Visual Geometry and a Visual 3D Landmark in Zebrafish (Danio rerio).

Fish conjoin environmental geometry with conspicuous landmarks to reorient towards foraging sites and social stimuli. Zebrafish (Danio rerio) can merge a rectangular opaque arena with a 2D landmark (a blue-colored wall) but cannot merge a rectangular transparent arena with a 3D landmark (a blue cylinder) without training to "feel" the environment thanks to other-than-sight pathways. Thus, their success is linked to tasks differences (spontaneous vs. rewarded). This study explored the reorientation behavior of zebrafish within a rectangular transparent arena, with a blue cylinder outside, proximal to/distal from a target corner position, on the short/long side of the arena. Adult males were extensively trained to distinguish the correct corner from the rotational one, sharing an equivalent metric-sense relationship (short surface left, long surface right), to access food and companions. Results showed that zebrafish's reorientation behavior was driven by both the non-visual geometry and the visual landmark, partially depending on the landmark's proximity and surface length. Better accuracy was attained when the landmark was proximal to the target corner. When long-term experience was allowed, zebrafish handled non-visual and visual sensory stimulations over time for reorienting. We advance the possibility that multisensory processes affect fish's reorientation behavior and spatial learning, providing a link through which to investigate animals' exploratory strategies to face situations of visual deprivation or impairments.

Quantity as a Fish Views It: Behavior and Neurobiology.

An ability to estimate quantities, such as the number of conspecifics or the size of a predator, has been reported in vertebrates. Fish, in particular zebrafish, may be instrumental in advancing the understanding of magnitude cognition. We review here the behavioral studies that have described the ecological relevance of quantity estimation in fish and the current status of the research aimed at investigating the neurobiological bases of these abilities. By combining behavioral methods with molecular genetics and calcium imaging, the involvement of the retina and the optic tectum has been documented for the estimation of continuous quantities in the larval and adult zebrafish brain, and the contributions of the thalamus and the dorsal-central pallium for discrete magnitude estimation in the adult zebrafish brain. Evidence for basic circuitry can now be complemented and extended to research that make use of transgenic lines to deepen our understanding of quantity cognition at genetic and molecular levels.

"Classifying-together" phenomenon in fish (Xenotoca eiseni): Simultaneous exposure to visual stimuli impairs subsequent discrimination learning.

When animals are previously exposed to two different visual stimuli simultaneously, their learning performance at discriminating those stimuli delays: such a phenomenon is known as "classifying-together" or "Bateson effect". However, the consistency of this phenomenon has not been wholly endorsed, especially considering the evidence collected in several vertebrates. The current study addressed whether a teleost fish, Xenotoca eiseni, was liable to the Bateson effect. Three experiments were designed, by handling the visual stimuli (i.e., a full red disk, an amputated red disk, a red cross) and the presence of an exposure phase, before performing a discriminative learning task (Exp. 1: full red disk vs. amputated red disk; Exp. 2: full red disk vs. red cross). In the exposure phase, three conditions per pairs of training stimuli were arranged: "congruence", where fish were exposed and trained to choose the same stimulus; "wide-incongruence", where fish were exposed to one stimulus and trained to choose the other one; "narrow-incongruence", where fish were exposed to both the stimuli and trained to choose one of them. In the absence of exposure (Exp. 3), the discrimination learning task was carried out to establish a baseline performance as regards the full red disk vs. amputated red disk, and the full red disk vs. red cross. Results showed that fish ran into retardation effects at learning when trained to choose a novel stimulus with respect to the one experienced during the exposure-phase (wide-incongruence condition), as well as after being simultaneously exposed to both stimuli (narrow-incongruence condition). Furthermore, there were no facilitation effects due to the congruence compared with the baseline: in such a case, familiar stimuli did not ease the performance at learning. The study provides the first evidence about the consistency of the classifying-together effect in a fish species, further highlighting the impact of visual similarities on discrimination processes.

The Geometric World of Fishes: A Synthesis on Spatial Reorientation in Teleosts.

Fishes navigate through underwater environments with remarkable spatial precision and memory. Freshwater and seawater species make use of several orientation strategies for adaptative behavior that is on par with terrestrial organisms, and research on cognitive mapping and landmark use in fish have shown that relational and associative spatial learning guide goal-directed navigation not only in terrestrial but also in aquatic habitats. In the past thirty years, researchers explored spatial cognition in fishes in relation to the use of environmental geometry, perhaps because of the scientific value to compare them with land-dwelling animals. Geometric navigation involves the encoding of macrostructural characteristics of space, which are based on the Euclidean concepts of "points", "surfaces", and "boundaries". The current review aims to inspect the extant literature on navigation by geometry in fishes, emphasizing both the recruitment of visual/extra-visual strategies and the nature of the behavioral task on orientation performance.

Visual discrimination and amodal completion in zebrafish.

While zebrafish represent an important model for the study of the visual system, visual perception in this species is still less investigated than in other teleost fish. In this work, we validated for zebrafish two versions of a visual discrimination learning task, which is based on the motivation to reach food and companions. Using this task, we investigated zebrafish ability to discriminate between two different shape pairs (i.e., disk vs. cross and full vs. amputated disk). Once zebrafish were successfully trained to discriminate a full from an amputated disk, we also tested their ability to visually complete partially occluded objects (amodal completion). After training, animals were presented with two amputated disks. In these test stimuli, another shape was either exactly juxtaposed or only placed close to the missing sectors of the disk. Only the former stimulus should elicit amodal completion. In human observers, this stimulus causes the impression that the other shape is occluding the missing sector of the disk, which is thus perceived as a complete, although partially hidden, disk. In line with our predictions, fish reinforced on the full disk chose the stimulus eliciting amodal completion, while fish reinforced on the amputated disk chose the other stimulus. This represents the first demonstration of amodal completion perception in zebrafish. Moreover, our results also indicated that a specific shape pair (disk vs. cross) might be particularly difficult to discriminate for this species, confirming previous reports obtained with different procedures.

Archerfish number discrimination.

Debates have arisen as to whether non-human animals actually can learn abstract non-symbolic numerousness or whether they always rely on some continuous physical aspect of the stimuli, covarying with number. Here, we investigated archerfish (Toxotes jaculatrix) non-symbolic numerical discrimination with accurate control for covarying continuous physical stimulus attributes. Archerfish were trained to select one of two groups of black dots (Exp. 1: 3 vs 6 elements; Exp. 2: 2 vs 3 elements); these were controlled for several combinations of physical variables (elements' size, overall area, overall perimeter, density, and sparsity), ensuring that only numerical information was available. Generalization tests with novel numerical comparisons (2 vs 3, 5 vs 8, and 6 vs 9 in Exp. 1; 3 vs 4, 3 vs 6 in Exp. 2) revealed choice for the largest or smallest numerical group according to the relative number that was rewarded at training. None of the continuous physical variables, including spatial frequency, were affecting archerfish performance. Results provide evidence that archerfish spontaneously use abstract relative numerical information for both small and large numbers when only numerical cues are available.

Towards a standardization of non-symbolic numerical experiments: GeNEsIS, a flexible and user-friendly tool to generate controlled stimuli.

Several studies have suggested that vertebrate and invertebrate species may possess a number sense, i.e. an ability to process in a non-symbolic and non-verbal way the numerousness of a set of items. However, this hypothesis has been challenged by the presence of other non-numerical continuous physical variables, which vary along with numerosity (i.e., any change in the number of visual physical elements in a set naturally involves a related change in visual features such as area, density, contour length and convex hull of the stimulus). It is therefore necessary to control and manipulate the continuous physical information when investigating the ability of humans and other animals to perceive numerousness. During decades of research, different methods have been implemented in order to address this issue, which has implications for experiment replicability and inter-species comparisons, since no general standardized procedure is currently being used. Here we present the 'Generation of Numerical Elements Images Software' (GeNEsIS) for the creation of non-symbolic numerical arrays in a standardized and user-friendly environment. The main aim of this tool is to provide researchers in the field of numerical cognition a manageable and precise instrument to produce visual numerical arrays controlled for all the continuous variables. Additionally, we implemented the ability to actively guide stimuli presentation during habituation/dishabituation and dual-choice comparison tasks used in human and comparative research.

Neurons in the Dorso-Central Division of Zebrafish Pallium Respond to Change in Visual Numerosity.

We found a region of the zebrafish pallium that shows selective activation upon change in the numerosity of visual stimuli. Zebrafish were habituated to sets of small dots that changed in individual size, position, and density, while maintaining their numerousness and overall surface. During dishabituation tests, zebrafish faced a change in number (with the same overall surface), in shape (with the same overall surface and number), or in size (with the same shape and number) of the dots, whereas, in a control group, zebrafish faced the same stimuli as during the habituation. Modulation of the expression of the immediate early genes c-fos and egr-1 and in situ hybridization revealed a selective activation of the caudal part of the dorso-central division of the zebrafish pallium upon change in numerosity. These findings support the existence of an evolutionarily conserved mechanism for approximate magnitude and provide an avenue for understanding its underlying molecular correlates.

The Sense of Number in Fish, with Particular Reference to Its Neurobiological Bases.

It is widely acknowledged that vertebrates can discriminate non-symbolic numerosity using an evolutionarily conserved system dubbed Approximate Number System (ANS). Two main approaches have been used to assess behaviourally numerosity in fish: spontaneous choice tests and operant training procedures. In the first, animals spontaneously choose between sets of biologically-relevant stimuli (e.g., conspecifics, food) differing in quantities (smaller or larger). In the second, animals are trained to associate a numerosity with a reward. Although the ability of fish to discriminate numerosity has been widely documented with these methods, the molecular bases of quantities estimation and ANS are largely unknown. Recently, we combined behavioral tasks with molecular biology assays (e.g c-fos and egr1 and other early genes expression) showing that the thalamus and the caudal region of dorso-central part of the telencephalon seem to be activated upon change in numerousness in visual stimuli. In contrast, the retina and the optic tectum mainly responded to changes in continuous magnitude such as stimulus size. We here provide a review and synthesis of these findings.

The geometry as an eyed fish feels it in spontaneous and rewarded spatial reorientation tasks.

Disoriented human beings and animals, the latter both sighted and blind, are able to use spatial geometric information (metric and sense properties) to guide their reorientation behaviour in a rectangular environment. Here we aimed to investigate reorientation spatial skills in three fish species (Danio rerio, Xenotoca eiseni, Carassius auratus) in an attempt to discover the possible involvement of extra-visual senses during geometric navigation. We observed the fish's behaviour under different experimental procedures (spontaneous social cued task and rewarded exit task), providing them different temporal opportunities to experience the environmental shape (no experience, short and prolonged experience). Results showed that by using spontaneous social cued memory tasks, fishes were not able to take advantage of extra-visual senses to encode the spatial geometry, neither allowing them short time-periods of environmental exploration. Contrariwise, by using a reference memory procedure, during the rewarded exit tasks, thus providing a prolonged extra-visual experience, fishes solved the geometric task, showing also differences in terms of learning times among species.

Response to change in the number of visual stimuli in zebrafish:A behavioural and molecular study.

Evidence has shown that a variety of vertebrates, including fish, can discriminate collections of visual items on the basis of their numerousness using an evolutionarily conserved system for approximating numerical magnitude (the so-called Approximate Number System, ANS). Here we combine a habituation/dishabituation behavioural task with molecular biology assays to start investigating the neural bases of the ANS in zebrafish. Separate groups of zebrafish underwent a habituation phase with a set of 3 or 9 small red dots, associated with a food reward. The dots changed in size, position and density from trial to trial but maintained their numerousness, and the overall areas of the stimuli was kept constant. During the subsequent dishabituation test, zebrafish faced a change (i) in number (from 3 to 9 or vice versa with the same overall surface), or (ii) in shape (with the same overall surface and number), or (iii) in size (with the same shape and number). A control group of zebrafish was shown the same stimuli as during the habituation. RT-qPCR revealed that the telencephalon and thalamus were characterized by the most consistent modulation of the expression of the immediate early genes c-fos and egr-1 upon change in numerousness; in contrast, the retina and optic tectum responded mainly to changes in stimulus size.

The Environmental Geometry in Spatial Learning by Zebrafish (Danio rerio).

During navigation, disoriented animals learn to use the spatial geometry of rectangular environments to gain rewards. The length of macroscopic surfaces (metric: short/long) and their spatial arrangement (sense: left/right) are powerful cues that animals prove to encode for reorientation. The aim of this study was to investigate if zebrafish (Danio rerio) could take advantage of such geometric properties in a rewarded exit task, by applying a reference memory procedure. The experiment was performed in a rectangular arena having four white walls, where fish were required to choose the two geometrically equivalent exit corners lying on the reinforced diagonal. Results showed that zebrafish encoded the geometry of the arena during reorientation, solving the spatial task within the first 5 days of training. With the aim to avoid the possible influence of extravisual cues on the zebrafish success, we performed a geometric test in extinction of response after the learning day. At test, fish persisted in choosing the two correct corners, thus confirming that the navigation strategy used at training was based on geometric cues. This study adds evidence about the role of geometric frameworks in fish species, and it further validates an effective spatial learning paradigm for zebrafish.

Use of numerical and spatial information in ordinal counting by zebrafish.

The use of non-symbolic numerical information is widespread throughout the animal kingdom, providing adaptive benefits in several ecological contexts. Here we provide the possible evidence of ordinal numerical skills in zebrafish (Danio rerio). Zebrafish were trained to identify the second exit in a series of five identically-spaced exits along a corridor. When at test the total length of the corridor (Exp. 1) or the distance between exits (Exp. 2) was changed, zebrafish appeared not to use the absolute spatial distance. However, zebrafish relied both on ordinal as well as spatial cues when the number of exits was increased (from 5 to 9) and the inter-exit distance was reduced (Exp. 3), suggesting that they also take into account relative spatial information. These results highlight that zebrafish may provide a useful model organism for the study of the genetic bases of non-symbolic numerical and spatial cognition, and of their interaction.

A Detour Task in Four Species of Fishes.

Four species of fish (Danio rerio, Xenotoca eiseni, Carassius auratus, and Pterophyllum scalare) were tested in a detour task requiring them to temporarily abandon the view of the goal-object (a group of conspecifics) to circumvent an obstacle. Fishes were placed in the middle of a corridor, at the end of which there was an opaque wall with a small window through which the goal was visible. Midline along the corridor two symmetrical apertures allowed animals to access two compartments for each aperture. After passing the aperture, fishes showed searching behavior in the two correct compartments close to the goal, appearing able to localize it, although they had to temporarily move away from the object's view. Here we provide the first evidence that fishes can solve such a detour task and therefore seem able to represent the "permanence in existence" of objects, which continue to exist even if they are not momentarily visible.

Extra-Visual Systems in the Spatial Reorientation of Cavefish.

Disoriented humans and animals are able to reorient themselves using environmental geometry ("metric properties" and "sense") and local features, also relating geometric to non-geometric information. Here we investigated the presence of these reorientation spatial skills in two species of blind cavefish (Astyanax mexicanus and Phreatichthys andruzzii), in order to understand the possible role of extra-visual senses in similar spatial tasks. In a rectangular apparatus, with all homogeneous walls (geometric condition) or in presence of a tactilely different wall (feature condition), cavefish were required to reorient themselves after passive disorientation. We provided the first evidence that blind cavefish, using extra-visual systems, were able i) to use geometric cues, provided by the shape of the tank, in order to recognize two geometric equivalent corners on the diagonal, and ii) to integrate the geometric information with the salient cue (wall with a different surface structure), in order to recover a specific corner. These findings suggest the ecological salience of the environmental geometry for spatial orientation in animals and, despite the different niches of adaptation, a potential shared background for spatial navigation. The geometric spatial encoding seems to constitute a common cognitive tool needed when the environment poses similar requirements to living organisms.

Quantity discrimination by zebrafish (Danio rerio).

Discrimination of quantity (magnitude) was investigated in zebrafish (Danio rerio). Male zebrafish chose to approach the location previously occupied by the larger in number between 2 groups of female conspecifics (no longer visible at test) in sets of 1 versus 2 items, and 2 versus 3 items, but failed at 3 versus 4 items; similarly, when tested with larger numbers, zebrafish succeeded with 2 versus 4, 4 versus 6, and 4 versus 8 items, but failed with 6 versus 8 items. The results suggest that zebrafish rely on an approximate number system to discriminate memorized sets of conspecifics of different magnitudes, the degree of precision in recall being mainly dependent on the ratio between the sets to be discriminated.

Learning of geometry and features in bumblebees (Bombus terrestris).

Bumblebees (Bombus terrestris) were trained to find one of the four exit holes located in the corners of an enclosed environment with a distinctive geometry (a rectangular cage). Panels located at the corners provided nongeometric, featural cues. Between trials bumblebees were passively disoriented to disable dead reckoning. When tested after removal of the panels, bumblebees reoriented using the residual information provided by the geometry of the cage. When tested after removal of only the two panels located in the two geometrically correct corners (the one with the exit and the diagonally opposite one), bumblebees were not able to use features in corners distant to the goal to reorient themselves. Finally, when geometric and featural cues provided contradictory information, bumblebees showed more reliance on featural cues. A similar outcome was observed when the conflict between geometrical and featural information was determined by first training bumblebees in a rectangular cage with a single wall of a different color used as a feature, and then testing animals with the feature displaced along a different wall. When the feature was close to the goal during training, bumblebees chose the corners with the feature at test, when the feature was far from goal during training, bumblebees chose the corners with the correct geometry at test. These results are similar to those revealed by similar transformational tests carried out in vertebrates relying mainly on vision for spatial orientation, that is, birds and monkeys.

Discrimination of small quantities by fish (redtail splitfin, Xenotoca eiseni).

Discrimination of quantity has been argued to rely on two non-verbal representational systems: an object file system (OFS) for representing small values (≤3-4) and an analog magnitude system (AMS) for representing large magnitudes (>4). Infants' ability to discriminate 1 versus 2, 1 versus 3, 2 versus 3, but not 1 versus 4 or 2 versus 4 seems to prove the independence of such systems. Here, we show that redtail splitfin fish (Xenotoca eiseni) performed relative quantity estimations preferring to approach the location previously occupied by the larger in number between two groups of conspecifics (no longer visible at test) in sets of 1 versus 2 and 2 versus 3 items, but failed at 3 versus 4 items, thus showing the same set-size limit as infants for discrimination of small quantities. However, when tested with quantities that spanned the boundary of the two systems, that is, 1 versus 4 and 2 versus 4, fish succeeded. These results thus point to either the use of continuous physical variables and/or the use of the AMS also for small numerousness in fish in this task.